Wt1 modified peptide

ABSTRACT

The present invention discloses a cancer antigen peptide comprising the following amino acid sequence: Cys Tyr Thr Trp Asn Gln Met Asn Leu (Sequence ID No. 3), a cancer vaccine having this for its active ingredient, and a DNA vaccine having for its active ingredient DNA that codes for this peptide.

TECHNICAL FIELD

[0001] The present invention relates to a cancer antigen based on theproduct of Wilms' tumor suppresser gene WT1. This cancer antigen isuseful as an anticancer vaccine against cancers of the blood such asleukemia, myelodysplastic syndrome, multiple myeloma and malignantlymphoma, solid cancers such as gastric cancer, colon cancer, lungcancer, breast cancer, germinal cell cancer, liver cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical carcinoma andovarian cancer, as well as any cancer that expresses WT1.

DESCRIPTION OF THE RELATED ART

[0002] The immune mechanism for eliminating foreign objects from thebody generally consists of humoral immunity, in which is involvedmacrophages that function as antigen-presenting cells that recognize anantigen, helper T-cells that activate other T-cells by recognizingantigens presented by said macrophages and producing variouslymphokines, and B lymphocytes that differentiate intoantibody-producing cells due to the action of said lymphokines; and,cellular immunity, by which killer T-cells (cytotoxic T-cells (CTL)),which have differentiated as a result of being presented with anantigen, attack and destroy target cells.

[0003] At present, cancer immunity is thought to mainly be the result ofcellular immunity involving killer T-cells. In cancer immunity affectedby killer T-cells, precursor T-cells, which have recognized cancerantigen presented in the form of a complex of major histocompatibilitycomplex (MHC) class I (MHC class I antigen, also referred to as HLAantigen in the case of humans) and cancer antigen, differentiate andproliferate, and the resulting killer T-cells that have formed attackand destroy the cancer cells. At this time, the cancer cells present acomplex of MHC class I antigen and cancer antigen on their cell surface,and this is targeted by the killer T-cells (Cur. Opin. Immunol., 5, 709,1993; Cur. Opin. Immunol., 5, 719, 1993; Cell, 82, 13, 1995; Immunol.Rev., 146, 167, 1995).

[0004] The aforementioned cancer antigen presented by MHC class Iantigen on the cancer cells serving as the target cells is thought to bea peptide composed of about 8-12 amino acids formed as a result ofantigen protein synthesized within cancer cells being processed byintracellular protease (Cur. Opin. Immunol., 5, 709, 1993; Cur. Opin.Immunol., 5, 719, 1993; Cell, 82, 13, 1995; Immunol. Rev., 146, 167,1995).

[0005] At present, although searches have been conducted for antigenproteins for various cancers, few have been verified to becancer-specific antigens.

[0006] The tumor suppresser gene WT1 of Wilms tumor (WT1 gene) has beenisolated from chromosome 11p13 as one of the causative genes of Wilmstumor based on analysis of the WAGR syndrome that occurs as acomplication of Wilms tumor, aniridia, urogenital abnormalities, mentalretardation and so forth (Gessler, M., et al., Nature, Vol. 343, p.774-778 (1990)). Its genomic DNA is about 50 kb and is composed of 10exons, while its cDNA is about 3 kb. The amino acid sequence estimatedfrom cDNA is as shown in Sequence ID No. 1 (Mol. Cell. Biol., 11, 1707,1991).

[0007] The WT1 gene is expressed with high frequency in human leukemia,and when leukemia cells are treated with WT1 antisense oligomer, thegrowth of the cells is inhibited (Japanese Unexamined Patent PublicationNo. 9-104627). Thus, WT1 gene is thought to act to promote the growth ofleukemia cells. Moreover, WT1 is also highly expressed in solid cancerssuch as gastric cancer, colon cancer, lung cancer, breast cancer,germinal cell cancer, liver cancer, skin cancer, bladder cancer,prostate cancer, uterine cancer, cervical carcinoma and ovarian cancer(Japanese Patent Application No. 9-191635), and the WT1 gene has beendemonstrated to be a novel tumor marker in leukemia and solid cancers.

[0008] Several cancer-specific antigen peptides consisting of a portionof the WT1 gene expression product are described in WO 00/06602, oneparticularly promising peptide is designated as D^(b), and the followingamino acid sequence: Cys Met Thr Trp Asn Gln Met Asn Leu (Sequence IDNo. 2) (referred to as “WT1 wild peptide” in the present invention) isdescribed therein.

DISCLOSURE OF THE INVENTION

[0009] Thus, an object of the present invention is to provide a peptidethat is promising as a cancer vaccine and which has higher activity thanpreviously known cancer-specific antigen peptides.

[0010] As a result of earnestly conducting various studies to solve theabove problems, the inventors of the present invention found that apeptide (referred to as “WT1 modified peptide”) having an amino acidsequence in which the second amino acid Met of the aforementioned knownamino acid sequence (Sequence ID No. 2) is changed to Tyr, namely CysTyr Thr Trp Asn Gln Met Asn Leu (Sequence ID No. 3), has higheractivity, thereby leading to completion of the present invention.

[0011] Thus, the present invention provides a peptide (WT1 modifiedpeptide) consisting of 9-30 amino acids and comprising the followingamino acid sequence: Cys Tyr Thr Trp Asn Gln Met Asn Leu (Sequence IDNo. 3). This peptide is preferably a polypeptide consisting of 9-12amino acids and comprising the amino acid sequence indicated in SequenceID No. 3 and, more preferably, a peptide consisting of the amino acidsequence indicated in Sequence ID No. 3.

[0012] Moreover, the present invention provides a cancer vaccine havingfor its active ingredient the aforementioned WT1 modified peptide.

[0013] Moreover, the present invention also provides a DNA vaccineagainst cancer having for its active ingredient DNA coding for theaforementioned peptide.

[0014] In addition, the present invention provides antigen-presentingcells on which presented a complex of HLA antigen (MHC class I antigen)and the aforementioned peptide.

[0015] Moreover, the present invention also provides cytotoxic T-cellsthat recognize a complex of HLA antigen and the aforementioned peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a graph showing the cell killing effects (specificcytolytic activity) on C1R2402 target cells (T), either pulsed or notpulsed with peptide, by effecter cells (E) stimulated with WT1 wildpeptide (Sequence ID No. 2) or the WT1 modified peptide of the presentinvention (Sequence ID No. 3). In the graph, the black circles indicatethe cytolytic effect on C1R2402 target cells pulsed with wild peptide byeffecter cells stimulated with WT1 modified peptide, the black squaresindicate the cytolytic effect on C1R2404 target cells pulsed with wildpeptide by effecter cells stimulated with WT1 wild peptide, the whitecircles indicate the cytolytic effect on C1R2402 target cells not pulsedwith wild peptide by effecter cells stimulated with WT1 modifiedpeptide, and the white squares indicate the cytolytic effect on C1R2402target cells not pulsed with wild peptide by effecter cells stimulatedwith WT1 wild peptide.

[0017]FIG. 2 is a graph showing the cytolytic activity on acutemyelocytic leukemia cells endogenously expressing WT1 antigen or onacute myelocytic leukemia cells not expressing WT1 antigen by effectercells stimulated with WT1 wild peptide or the WT1 modified peptide ofthe present invention.

[0018]FIG. 3 is a graph showing the cell killing effects (specificcytolytic activity) on C1R2402 target cells, either pulsed or not pulsedwith peptide, by effecter cells stimulated with WT1 wild peptide or theWT1 modified peptide of the present invention. In the graph, the blackcircles indicate the cytolytic effect on C1R2402 cells pulsed with wildpeptide by effecter cells stimulated with WT1 modified peptide, theblack squares indicate the cytolytic effect on C1R2402 target cellspulsed with wild peptide by effecter cells stimulated with WT1 wildpeptide, the white circles indicate the cytolytic effect on C1R2402target cells not pulsed with wild peptide by effecter cells stimulatedwith WT1 modified peptide, and the white squares indicate the cytolyticeffect on C1R2402 target cells not pulsed with wild peptide by effectercells stimulated with WT1 wild peptide.

[0019]FIG. 4 is a graph showing the cytolytic activity on lung cancercell lines endogenously expressing WT1 or not expressing WT1 by effectercells stimulated with WT1 wild peptide or the WT1 modified peptide ofthe present invention.

[0020]FIG. 5 is a graph showing the inhibitory effects of anti-HLA classI antibody, anti-HLA class II antibody and anti-CD8 antibody on the cellkilling effects (specific cytolytic activity) on C1R2402 target cellspulsed with wild peptide by effecter cells stimulated by WT1 wildpeptide or the WT1 modified peptide of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The peptide of the present invention is a peptide consisting of9-30 amino acids that comprises the amino acid sequence consisting ofthe 9 amino acids shown in Sequence ID No. 3. Moreover, from theviewpoint of being presented by binding to HLA antigen, the peptide ispreferably a peptide consisting of 9-12 amino acids that comprises theamino acid sequence shown in Sequence ID No. 3 and, more preferably, isa peptide having rules (motifs) in the sequence of antigen peptidepresented by binding to HLA antigen at that time (J. Immunol., 152, p.3913, 1994; Immunogenetics, 41, p. 178, 1995; J. Immunol., 155, p. 4307,1994; J. Immunol., 155, p. 4749, 1995). Moreover, the peptide is mostpreferably a peptide consisting of an amino acid sequence of the 9 aminoacids shown in Sequence ID No. 3.

[0022] Furthermore, the aforementioned “peptide comprising the aminoacid sequence shown in Sequence ID No. 3” is specifically, for example,a peptide comprising the amino acid sequence shown in Sequence ID No. 3and extending in the direction of the N-terminal and/or the direction ofthe C-terminal from the applicable position on WT1 (Sequence ID No. 1)(position nos. 235-243) or from the corresponding position on human WT1(NCBI Database Accession No. XP012009), that has activity as a cancerantigen peptide.

[0023] An example of a method for measuring the activity of the cancerantigen peptide of the present invention is the method described in J.Immunol., 154, p. 2257, 1995. The following provides an explanation ofan outline of this method using the case of the type of HLA beingHLA-A24 as an example. First, peripheral blood lymphocytes are isolatedfrom a person positive for HLA-A24 antigen. Next, by stimulating theperipheral blood lymphocytes by adding the peptide of the presentinvention in vitro, CTL (cytotoxic T-cells) are induced thatspecifically recognize the complex of the peptide of the presentinvention and HLA-A24 presented by the antigen-presenting cells.

[0024] This induction of CTL can be investigated by, for example,measuring the amounts of various cytokines (e.g., IFN-γ) produced by theCTL by reacting with the complex of antigen peptide and HLA-A24. Inaddition, induction of CTL can also be investigated by a method in whichthe cytotoxicity of the CTL is measured with respect to antigenpeptide-presenting cells labeled with ⁵¹Cr or Europium (⁵¹Cr ReleaseAssay, Int. J. Cancer, 58, p. 317, 1994; Europium Release Assay, J.Immunol., 154, p. 3991, 1995). Moreover, induction of CTL can also beinvestigated by referring to the examples described later.

[0025] The present invention also relates to a cancer vaccine that hasthe aforementioned antigen as its active ingredient. This vaccine can beused for the prevention or treatment of cancers of the blood such asleukemia, myelodysplastic syndrome, multiple myeloma and malignantlymphoma, as well as solid cancers such as gastric cancer, colon cancer,lung cancer, breast cancer, germinal cell cancer, liver cancer, skincancer, bladder cancer, prostate cancer, uterine cancer, cervicalcarcinoma and ovarian cancer. In particular, this vaccine can be appliedto patients positive for HLA-A24. This vaccine can be administeredorally or parenterally by for example, intraperitoneal, subcutaneous,intracutaneous, intramuscular, intravenous or intranasal administration.

[0026] Moreover, administration of the vaccine of the present inventioncan also be carried out by a method in which monocytes are collectedfrom the peripheral blood of a patient, dendritic cells are extractedfrom the monocytes, the dendritic cells are pulsed with the peptide ofthe present invention and then returned to the patient by subcutaneousadministration and so forth.

[0027] This method is referred to as cytotherapy or dendritic cell (DC)therapy, and the section entitled “Antigen-Presenting Cells” describedlater should be referred to for further details.

[0028] The vaccine, in addition to the peptide administered as theaforementioned active ingredient, may also contain pharmaceuticallyallowable carriers such as a suitable adjuvant (Clin-Microbiol. Rev., 7,277-289, 1994), examples of which include a mineral gel like aluminumhydroxide, a surfactant like phosphorous lecithin and a pluronicpolyole, a polyanion, a peptide and an oily emulsion. Alternatively, thevaccine may contain other aggregates mixed into liposomes or blendedinto polysaccharide or the vaccine. The dosage is typically 0.1 μg/kg to1 mg/kg per day.

[0029] In the present invention, DNA that codes the aforementionedpolypeptide vaccine can also be used as a vaccine (DNA vaccine). Namely,after inserting nucleic acids, and preferably DNA, that contain nucleicacids that encode the WT1 modified peptide of the present invention intoa suitable vector, and preferably an expression vector, cancer immunitycan be imparted by administering the vector to an animal. WO 00/6602 orJ. Immunol., 160, p. 1717, 1998 and so forth should be referred to forthe specific technique used for this DNA vaccine.

[0030] In addition, the present invention relates to antigen-presentingcells on which a complex of HLA antigen and the aforementioned peptideis presented. In this example, although potent cell killing activity isobserved due to stimulation with the peptide of the present invention,this is the result of the presence of antigen-presenting cells, on whicha complex of the peptide of the present invention and HLA antigen(HLA-A24 antigen) is presented, within peripheral blood monocytes, andof the induction of CTL (cytotoxic T-cells) that specifically recognizethese antigen-presenting cells. These antigen-presenting cells on whicha complex of HLA antigen and the peptide of the present invention ispresented are used effectively in cytotherapy (DC therapy) as describedbelow.

[0031] The antigen-presenting cells used in cytotherapy are produced byisolating cells having the ability to present antigen from tumorpatients, pulsing these cells with peptide of the present inventionoutside the body, and causing a complex of HLA antigen and the peptideof the present invention to be presented on the surface of the cells.Here, although there are no particular restrictions on the “cells havingthe ability to present antigen” provided they are cells that express HLAantigen capable of presenting the peptide of the present invention onthe surface of the cells, dendritic cells are preferable since they areconsidered to have high antigen-presenting ability.

[0032] In addition, the peptide of the present invention that is used topulse the aforementioned cells having the ability to present antigen maynot only be in the form of a peptide, but rather may also be in the formof DNA or RNA that encodes said peptide.

[0033] A specific method for preparing the antigen-presenting cells ofthe present invention can be referred to in, for example, CancerImmunol. Immunother., 46, 82, 1998, J. Immunol., 158, p. 1796, 1997, andCancer Res., 59, p. 1184, 1999. In the case of using dendritic cells,lymphocytes are isolated from the peripheral blood of a tumor patientsusing the Fycoll method, and after subsequently removing the non-adheredcells, dendritic cells are derived from the adhered cells by culturingin the presence of GM-CSF and IL-4, after which the antigen-presentingcells of the present invention can be prepared by pulsing the dendriticcells by culturing with the peptide of the present invention.

[0034] In addition, in the case of preparing the antigen-presentingcells of the present invention by inserting DNA or RNA encoding thepeptide of the present invention into the aforementioned cells havingthe ability to present antigen, insertion can be carried out byreferring to, for example, Cancer Res., 56, p. 5672, 1996 or J.Immunol., 161, p. 5607, 1998 in the case of DNA, or by referring to J.Exp. Med., 184, p. 465, 1996 in the case of RNA.

[0035] These antigen-presenting cells can be used as the activeingredient of a tumor therapeutic agent. At that time, in order tomaintain the stability of the antigen-presenting cells, the treatmentagent preferably comprises physiological saline, phosphate-bufferedsaline (PBS) or medium and so forth. Examples of administration methodsinclude intravenous administration, subcutaneous administration andintracutaneous administration.

[0036] Moreover, the present invention also relates to cytotoxic T-cells(CTL) that recognize a complex of HLA antigen and the aforementionedpeptide. The CTL of the present invention can be effectively used in theadoptive immunotherapy described below.

[0037] Namely, in the case of melanoma, adoptive immunotherapy has beenrecognized to be therapeutically effective by culturing a large numberof the patient's T-cells that have invaded the tumor in vitro, and thenreturning them to the patient (J. Natl. Cancer Inst., 86, 1159, 1994).In addition, in the case of mouse melanoma, inhibition of metastasis hasbeen observed by stimulating spleen cells in vitro with tumor antigenpeptide TRP-2, allowing specific CTL to proliferate in the tumor antigenpeptide, and then administering said CTL to melanoma-transplanted mice(J. Exp. Med., 185, 453, 1997). This is based on the result of allowingCTL to proliferate in vitro that specifically recognize a complex of theHLA antigen of antigen-presenting cells and tumor antigen peptide. Thus,a treatment method in which patient peripheral blood lymphocytes arestimulated in vitro using the peptide of the present invention toincrease tumor-specific CTL followed by returning these cells to thepatient is thought to be useful.

[0038] In this manner, the CTL of the present invention can be used asthe active ingredient of a tumor therapeutic agent. At that time, inorder to maintain the stability of the CTL, the therapeutic agentpreferably comprises physiological saline, phosphate-buffered saline(PBS) or medium and so forth. Examples of administration methods includeintravenous administration, subcutaneous administration andintracutaneous administration.

[0039] The following examples serve to clarify the usefulness of thepeptide of the present invention as a cancer antigen and cancer vaccine.

EXAMPLE 1

[0040] Peripheral blood mononuclear cells were isolated fromHLA-A*2402-positive donors and distributed among the wells of a 24-wellplate at 2×10⁶ cells/well followed by the addition of WT1 wild peptideor WT1 modified peptide to a concentration of 20 μM and culturing for 1week. The medium used at this time consisted of 45% RPMI, 45% AIV, 10%FCS 1× non-essential amino acids and SM/PCG. Following theaforementioned culturing, the cells were adjusted to 2×10⁶ cells/welland used as responder cells.

[0041] On the other hand, other peripheral blood mononuclear cells weresimilarly isolated from the same HLA-A*2402-positive donors and thenpeptide-pulsed by culturing for 4 days with one of the aforementionedpeptides at 20 μM. After then irradiating at 30 Gy, the cells wereadjusted to 4×10⁶ cells/well and used as stimulator cells.

[0042] The responder cells and stimulator cells prepared in the mannerdescribed above were then mixed and then cultured for 1 week followingthe addition of IL-2 at 50 U/ml. As a result, the status of theresulting cells were as shown in the following table. TABLE 1 PeptideNo. of cells CD4 CD8 WT1 wild peptide 2.4 × 10⁶/well  5% 35% WT1modified 3.0 × 10⁶/well 18% 38% peptide

[0043] Next, a killing assay was carried out in accordance with ⁵¹Crrelease method (J. Immunol., 164, 1873, 2000). C1R2402 cells and C1R2402cells pulsed with the aforementioned peptides were used for the targetcells. Cells stimulated by WT1 wild peptide or WT1 modified peptide aspreviously described (effector cells (E)) were then allowed to act oneach of these target cells (T) at an E:T ratio of 1, 5 of 20, followedby measurement of cell lysis. Those results are shown in FIG. 1. As isclear from this graph, cells stimulated with WT1 modified peptideexhibited a more potent cell killing activity than cells stimulated withWT1 wild peptide.

EXAMPLE 2

[0044] The cell killing activity of effector cells stimulated with WT1wild peptide or WT1 modified peptide on leukemia cells that endogenouslyexpress WT1 antigen was tested according to the ⁵¹Cr release method.WT1+/A*2402+ cells (leukemia cells from AML patient #1), WT1−/A*2402+cells (leukemia cells from AML patient #2), WT1+/A*2402− cells (leukemiacells from AML patient #3) and WT1−/A*2402− cells (leukemia cells fromAML patient #4) were used for the target cells.

[0045] The effector cells (E) prepared in Example 1 and theaforementioned target cells (T) were mixed at an E:T ratio of 20:1 andcultured for 4 hours followed by measurement of the degree of celllysis. Those results are shown in FIG. 2.

[0046] As is clear from this graph, although both the cells stimulatedwith WT1 wild peptide or WT1 modified peptide demonstrated cytotoxicactivity on the WT1+/A*2402 cells, the level of that activity was higherfor the WT1 modified peptide.

EXAMPLE 3

[0047] The same experiment as Example 1 was carried out using effectorcells prepared from peripheral blood mononuclear cells of differenthealthy donors positive for HLA-A*2402. Those results are shown in FIG.3.

[0048] As is clear from this graph, similar to Example 1, cellsstimulated with WT1 modified peptide exhibited a more potent cytotoxicactivity than cells stimulated with WT1 wild peptide.

EXAMPLE 4

[0049] The cytotoxic activity of effector cells stimulated with WT1 wildpeptide or WT1 modified peptide was tested on a cancer cell lineassociated with lung cancer that endogenously expresses WT1 antigen(target cells) using the ⁵¹Cr release method. RERF-LCAI (WT1+/A*2402+),LC1sq (WT1+/A*2402+), 11-18 (WT1−/A*2402+) and LK87 (WT1+/A*2402−) cellswere used for the target cells.

[0050] Effector cells (E) prepared in the same manner as Example 1 andthe aforementioned target cells (T) labeled with ⁵¹Cr were cultured for4 hours at an E:T ratio of 20:1 in the same manner as Example 2 followedby measurement of the degree of cell lysis. Those results are shown inFIG. 4.

[0051] As is clear from this graph, although both the cells stimulatedwith WT1 wild peptide or WT1 modified peptide demonstrated cytotoxicactivity only on the WT1+/A*2402+ cells, the level of that activity washigher for WT1 modified peptide.

EXAMPLE 5

[0052] Effector cells stimulated with WT1 wild peptide or WT1 modifiedpeptide were confirmed to be CD8-positive killer cells that bind to HLAclass I by a blocking assay using antibody. The antibodies usedconsisted of anti-HLA class I antibody, anti-HLA class II antibody andanti-CD8 antibody. Effector cells (E) prepared in the same manner asExample 1 and target cells (T) in the form of C1R2402 cells or C1R2402cells pulsed with WT1 wild peptide, both labeled with ⁵¹Cr, were mixedwith antibody at an E:T ratio of 20:1 and then cultured for 4 hoursfollowed by measurement of the degree of cell lysis according to the⁵¹Cr release method. Those results are shown in FIG. 5.

[0053] As is clear from this graph, cytotoxic activity was blocked byanti-HLA class I antibody and anti-CD8 antibody for both the cellsstimulated with WT1 wild peptide or WT1 modified peptide, indicatingthat the cells that exhibit cytotoxic activity are CD8-positive killercells that bind to HLA class I.

EXAMPLE 6

[0054] The binding affinity of WT1 modified peptide and WT1 wild peptideto HLA-A*2402 was investigated. After treating C1RA2402 cells for 1minute with a buffer solution (131 mM citric acid, 66 mM sodiumphosphate, 290 m osmol, pH 3.3), the cells were neutralized by addingDMEM medium comprising 0.5% bovine serum albumin. After washing thecells with the medium, they were suspended at a concentration of 2×10⁶cells/ml in DMEM medium containing 200 nM β2-microglobulin (Sigma) and0.5% bovine serum albumin. 15 μl of the cell suspension were mixed with50 μl of the medium comprising various concentrations of WT1 peptidefollowed by incubating for 4 hours at room temperature. After washingthe cells, they were stained with monoclonal antibody to HLA-A24 labeledwith FITC (clone name: 7A12), and the amount of HLA-A24 expressed wasanalyzed with a flow cytometer FACS system. A similar procedure wasperformed on the antigen peptide of melanoma antigen pmel 15, which hasbeen reported to bind to HLA-A*2402 (Ala Tyr Gly Leu Asp Phe Tyr IleLeu) (Sequence ID No. 4) (J. Immunol., 154, 5994, 1995), and using thisas a standard, the dissociation constants (Kd) of the WT1 peptides werecalculated according to the method described in the literature(Immunogenetics, 51, 816, 2000). These results are shown in Table 2.TABLE 2 Peptide Dissociation constant Kd (M) WT1 wild peptide 1.82 ×10⁻⁵ WT1 modified peptide 6.40 × 10⁻⁷

[0055] As is clear from this table, WT1 modified peptide demonstratedstronger binding affinity for HLA-A*2402 than the WT1 wild peptide.

[0056] On the basis of the aforementioned results, the peptide of thepresent invention was proven to unquestionably function as a cancerantigen, and cause the induction and proliferation of killer T-cells(cancer cell cytotoxic T-cells) against cancer cells. Thus, the cancerantigen peptide of the present invention is useful as a cancer vaccineagainst leukemia and solid cancers accompanying an increased expressionof the WT1 gene.

1 4 1 449 PRT Mouse 1 Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu LeuPro Ala Val Ser 5 10 15 Ser Leu Gly Gly Gly Gly Gly Gly Cys Gly Leu ProVal Ser Gly Ala 20 25 30 Arg Gln Trp Ala Pro Val Leu Asp Phe Ala Pro ProGly Ala Ser Ala 35 40 45 Tyr Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro AlaPro Pro Pro Pro 50 55 60 Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu ProSer Trp Gly Gly 65 70 75 80 Ala Glu Pro His Glu Glu Gln Cys Leu Ser AlaPhe Thr Leu His Phe 85 90 95 Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala CysArg Tyr Gly Pro Phe 100 105 110 Gly Pro Pro Pro Pro Ser Gln Ala Ser SerGly Gln Ala Arg Met Phe 115 120 125 Pro Asn Ala Pro Tyr Leu Pro Ser CysLeu Glu Ser Gln Pro Thr Ile 130 135 140 Arg Asn Gln Gly Tyr Ser Thr ValThr Phe Asp Gly Ala Pro Ser Tyr 145 150 155 160 Gly His Thr Pro Ser HisHis Ala Ala Gln Phe Pro Gln His Ser Phe 165 170 175 Lys His Glu Asp ProMet Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln 180 185 190 Tyr Ser Val ProPro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser 195 200 205 Cys Thr GlySer Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp 210 215 220 Asn LeuTyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln 225 230 235 240Met Asn Leu Gly Ala Thr Leu Lys Gly Met Ala Ala Gly Ser Ser Ser 245 250255 Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Gly Ile Gly Tyr Glu 260265 270 Ser Glu Asn His Thr Ala Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile275 280 285 His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg ValSer 290 295 300 Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr SerGlu Lys 305 310 315 320 Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn LysArg Tyr Phe Lys 325 330 335 Leu Ser His Leu Gln Met His Ser Arg Lys HisThr Gly Glu Lys Pro 340 345 350 Tyr Gln Cys Asp Phe Lys Asp Cys Glu ArgArg Phe Ser Arg Ser Asp 355 360 365 Gln Leu Lys Arg His Gln Arg Arg HisThr Gly Val Lys Pro Phe Gln 370 375 380 Cys Lys Thr Cys Gln Arg Lys PheSer Arg Ser Asp His Leu Lys Thr 385 390 395 400 His Thr Arg Thr His ThrGly Lys Thr Ser Glu Lys Pro Phe Ser Cys 405 410 415 Arg Trp His Ser CysGln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val 420 425 430 Arg His His AsnMet His Gln Arg Asn Met Thr Lys Leu His Val Ala 435 440 445 Leu 449 2 9PRT Artificial Sequence Synthetic Peptide 2 Cys Met Thr Trp Asn Gln MetAsn Leu 1 5 3 9 PRT Artificial Sequence Synthetic Peptide 3 Cys Tyr ThrTrp Asn Gln Met Asn Leu 1 5 4 9 PRT Artificial Sequence AntigenicPeptide 4 Ala Tyr Gly Leu Asp Phe Tyr Ile Leu 1 5

1. A cancer antigen peptide having for its active ingredient a peptideconsisting of 9-30 amino acids and comprising the following amino acidsequence: Cys Tyr Thr Trp Asn Gln Met Asn Leu (Sequence ID No. 3).
 2. Acancer antigen peptide according to claim 1 that is consisted of 9-12amino acids and comprises the amino acid sequence indicated in SequenceID No.
 3. 3. A cancer antigen peptide according to claim 1 that isconsisted of the amino acid sequence indicated in Sequence ID No.
 3. 4.A cancer vaccine having for its active ingredient a peptide according toany of claims 1 through
 3. 5. A DNA vaccine against cancer having forits active ingredient DNA that codes for a peptide according to any ofclaims 1 through
 3. 6. Antigen-presenting cells on which are presented acomplex of HLA antigen and a peptide according to any of claims 1through
 3. 7. Cytotoxic T-cells that recognize a complex of HLA antigenand a peptide according to any of claims 1 through 3.